Fungi are ubiquitous opportunistic parasites that infect a variety of species, including humans. Microsporidia, a phylum of unicellular fungi that include over 1200 species, infect a range of hosts. Species of the genus Encephalitozoon can disseminate in the host body and infect organs such as intestine, lung, kidney, brain, sinuses, muscles and eyes, and shed spores in feces, tissue, saliva, ocular and nasal discharged fluids, urine, mucus and blood. Microsporidiosis (i.e. microsporidia infection) causes chronic, debilitating diseases, including chronic and profuse diarrhea, electrolyte imbalance, food malabsorption, wasting syndrome, pulmonary microsporidiosis, respiratory failure, and even death. While microsporidia can infect healthy individuals, severe syndromes of microsporidiosis primarily occur in patients with compromised or suppressed immune-system, such as in senior citizens, HIV-infected individuals, cancer patients and organ transplant recipients.
Despite its adverse public health impacts, there lacks an accurate, sensitive, and convenient clinical assay capable of detecting microsporidia infection. As a result, a myriad of diseases associated with microsporidia remain vastly under-diagnosed. Common clinical assays include serological tests, transmission electron microscopy, histochemical methods, immunofluorescence, and polymerase chain reaction (PCR); however, all of them exhibit significant shortcomings. Serological tests produce high false-positive results in immuno-deficient individuals, who are often incapable of eliciting adequate and specific immune responses. Histochemical staining methods also produce high false-positive results in detecting microsporidia infection, since the commonly used dyes such as Calcofluor white, Fungi-fluor and Uvitex 2B stain a variety of fungi species indiscriminately. While transmission electron microscopy may accurately detect microsporidia, it is time-consuming and thus not practical for routine clinical uses. Similarly, immunofluorescence and PCR require special equipment and trained personnel, and thus, have limited applications beyond most research laboratories.
In addition, there is a lack of efficient techniques for detection or removal of microsporidia spores from environmental samples. For instance, in a publication by Hoffman R. M. et al., it was shown that continuous flow centrifugation only recovered 38.7-75.5% of microsporidia Encephalitozoon intestinalis seeded in filtered tap water. While immunomagnetic bead separation (IMS) enables a higher recovery of 78.8-90.2%, such recovery can be only obtained from certain environmental samples such as seeded ultrapure water samples, but can not be obtained from turbid samples. In addition, as particles that may interfere with PCR commonly exist in environmental samples and fungal spores, the combination of IMS and real-time PCR only detected six of the ten natural water samples seeded with 50 spores per L. Therefore, improved techniques for detection, isolation and/or removal of microsporidia as well as other pathogenic fungi species are urgently needed.